Despite great advances in AIDS diagnosis and treatment, the continuing devastation of the AIDS epidemic demands continuing efforts to understand all aspects of HIV replication, and to develop new methods for its inhibition. In pursuit of these goals, we have sought to define the activities of the HIV-1 structural (Gag) proteins so as to design antivirals that interfere with these functions. The Gag proteins are attractive targets since they perform multiple roles during the life cycle. The proteins initially are synthesized as N-terminally myristylated precursor (PrGag) proteins that employ their N-terminal matrix (MA) domains to target delivery to plasma membrane (PM) virus assembly sites. Evidence indicates that MA preferentially binds to the signaling phospholipid phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2), and that HIV-1 virus membranes are enriched for lipid raft constituents such as cholesterol, sphingomyelin, and ceramide. MA also has been shown to mediate the incorporation of the HIV-1 envelope (Env) glycoprotein complex into virus particles, and interacts with the cytoplasmic tail (CT) of the transmembrane (TM, gp41) portion of Env. Retrovirus matrix proteins also have been known to bind nucleic acids, and we recently discovered that the RNA and PI(4,5)P2 binding sites on MA overlap, supporting a new model in which RNA binding protects MA from association with inappropriate cellular membranes prior to PrGag delivery to the PM. Using our previous studies and preliminary results as a foundation, we propose novel approaches to dissect the mechanisms of matrix protein membrane, nucleic acid, and envelope protein binding, and to characterize methods for their inhibition. Our results will help elucidate how the HIV assembly machinery operates; and will lead to the development of Gag-targeted antivirals, and an understanding of how they work. To achieve these ends, our specific aims are as follows: 1. Characterization of the nucleic acid binding activity of the HIV-1 matrix protein. 2. Determination of membrane binding properties of HIV-1 MA. 3. Elucidation of HIV-1 matrix-envelope protein interactions. PUBLIC HEALTH RELEVANCE: Our investigations focus on an analysis of the activities of the HIV-1 precursor Gag (PrGag) protein, the structural protein of HIV. We propose to characterize the functions of the matrix (MA) Gag protein and how they may be inhibited. These studies are of direct public health relevance in that they will lead to the development of new Gag- targeted anti-HIV therapeutics and an understanding of how they work.